Various Bacillus and Paenibacillus Spp. Isolated From Soil Produce Compounds With Potent Antimicrobial Activity Against Clinically Relevant Pathogens

Abstract

The increasing prevalence of antibiotic resistance among clinically significant pathogens necessitates the discovery of novel antimicrobial agents. This study investigated 29 Bacillus and Paenibacillus isolates from the soil for antimicrobial activity against multidrug-resistant clinical pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Enterobacterales (CRE). In both agar- and broth-based antimicrobial assays, Paenibacillus profundus strains 7.5 and M4.5 exhibited potent broad-spectrum activity, including significant inhibition of many CREs. Species identification was performed through 16S rRNA sequencing, and genome mining of three producer strains using antiSMASH revealed biosynthetic gene clusters associated with a variety of nonribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs), and ribosomally synthesized and post-translationally modified peptides (RiPPs). While many of these clusters were not associated with known antimicrobial compounds, several of them displayed high similarity to known compounds such as polymyxin B, paenilan, colistin, and paenibacterin. These findings reinforce numerous previous studies highlighting the potential of soil-derived Bacillus and Paenibacillus species as valuable sources of novel antimicrobials to address the global antibiotic resistance crisis.

Keywords: Paenibacillus profundus; antimicrobial compounds; antimicrobial resistance; carbapenem‐resistant Enterobacterales; genome mining; soil bacteria.

Figure 1

Mechanisms of antibiotic resistance in bacteria. Bacteria have evolved many different strategies to protect themselves from the effects of antibiotics. Permeability barriers are intrinsic to the structure of the bacteria and provide resistance to specific groups of antibiotics. Porins in the membrane can be downregulated to decrease drug permeability. Some bacteria carry genes that encode enzymes that inactivate antibiotics or alter their targets, as well as increase drug efflux. Decreased metabolic activity is an example of a nongenetic mechanism of resistance.

Figure 2

Example of the cross‐streak method. A single colony of the producer strain (P. amylolyticus 9.5) is inoculated as a single vertical line on the plate. After incubating the growth line for 48 h, overnight cultures of the target strains (S. aureus, E. coli, P. aeruginosa, MRSA #1‐5) are streaked perpendicular to the producer strain. The antimicrobial activity of the producer strain against the target strain is measured in mm after 24 and 48 h of incubation.

Figure 3

PCR amplification of 16S rRNA gene fragments from representative bacterial isolates. The predicted size of each producer strain amplicon was slightly over 1500 bp, consistent with the ~1550 bp length of the 16S rRNA gene sequence. 1: ladder; 2: Aneurinibacillus migulanus 3.3; 3: Paenibacillus polymyxa 9.3 C; 4: Paenibacillus polymyxa 9.3 W; 5: Bacillus mojavensis 10.4. Similar amplicons were obtained with other selected isolates.

Figure 4

Diluted soil sample spread plate. Soil samples were diluted and spread on LB agar plates. Colonies with unique morphology were picked and tested for antimicrobial activity.

Figure 5

Colony morphologies of selected antimicrobial‐producing strains isolated from the soil around BYU campus. Isolates were grown at 30°C for 48‐72 h. 1: Bacillus mojavensis II; 2: Bacillus cereus 22; 3: Paenibacillus polymyxa 4.2; 4: Bacillus subtilis 16.2; 5: Aneurinibacillus migulanus 3.3; 6: Paenibacillus polymyxa 9.3 C; 7: Paenibacillus polymyxa 9.3 W; 8: Bacillus mojavensis 10.4; 9: Paenibacillus profundus 7.5; 10: Paenibacillus amylolyticus 9.5; 11: Paenibacillus amylolyticus 5.6; 12: Paenibacillus dendritiformis 6.7; 13: Paenibacillus dendritiformis 18.7; 14: Bacillus velezensis 3.8; 15: Bacillus halotolerans 5.9; 16: Bacillus halotolerans 8.9; 17: Paenibacillus alvei M13; 18: Bacillus mycoides M3.14; 19: Bacillus subtilis H18.1; 20: Paenibacillus profundus M4.5; 21: Paenibacillus dendritiformis M4.11; 22: Bacillus subtilis Y4B19; 23: Bacillus subtilis Y20; 24: Bacillus pumilus B3; 25: Bacillus safensis 17; 26: Bacillus mojavensis M4.2; 27: Bacillus pumilus Y3B7; 28: Bacillus pumilus 10.9; 29: Bacillus pumilus M2.12.

Figure 6

Phylogenetic tree showing the positions of the Bacillus and Paenibacillus producer strains relative to other members of those genera. Sequences were aligned using MEGA12, and evolutionary distances were computed using the Maximum Composite Likelihood method.

Figure 7

Inhibitory activity of producer strain cell‐free supernatants against target strains. Growth curves (left) and fold changes in growth (right) of: (A) Staphylococcus aureus. (B) Escherichia coli. (C) Pseudomonas aeruginosa. Growth curves depict 24 h of incubation with producer strain supernatants, while fold changes represent hour 24 of that growth period. Data shown as mean ± SD from three technical replicates. Statistical analysis was performed using two‐way ANOVA with Dunnett's multiple comparisons test to compare each treatment group to the untreated control at each time point. ns, not significant; *, **, ***, **** denote p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

Figure 8

Inhibitory activity of producer strain cell‐free supernatant against CRE target strains. Growth curves (left) and fold changes in growth (right) of: (A) CRE #1 Escherichia coli. (B) CRE #2 Enterobacter cloacae. (C) CRE #3 Klebsiella pneumoniae. (D) CRE #21 Citrobacter freundii. (E) CRE #26 Providencia stuartii. (F) CRE #27 Serratia marcescens. (G) CRE #29 Proteus mirabilis. (H) CRE #30 Shigella sonnei. (I) CRE #31 Salmonella Typhimurium. (J) CRE #56 Acinetobacter baumannii. (K) CRE #57 Morganella morganii. (L) CRE #134 Raoultella ornithinolytica. Growth curves depict 24 h of incubation with producer strain supernatants, while fold changes represent hour 24 of that growth period. Data shown as mean ± SD from three technical replicates. Statistical analysis was performed using two‐way ANOVA with Dunnett's multiple comparisons test to compare each treatment group to the untreated control at each time point. ns, not significant; *, **, ***, **** denote p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.